This project will examine the role of plasma hemoglobin in altered vascular responses observed in Sickle Cell Disease (SCO). Red cell lysis leads to increases in plasma hemoglobin levels that participate in vascular dysfunction, and such processes may lead to vascular complications of SCO such as pulmonary hypertension. Mechanisms for hemoglobin-mediated vascular dysfunction include nitric oxide (NO) scavenging by oxyhemoglobin and lipid oxidation by methemoglobin, both of which are present in plasma from SCO patients. The long term objective of this proposal is to understand the role of hemolysis in the pathology of SCO by examining the interplay between NO, lipid oxidation and hemolysis. We hypothesize that hemolysis will mediate vascular dysfunction through both NO and lipid-oxidation pathways. This will be addressed in four Specific Aims: 1) Examine in detail the fate of hemoglobin and heme in normal plasma and plasma from individuals with SCO. Examine the oxidation state and oxidizability of plasma from normals and individuals with SCO both in crisis and at a one month follow-up. 2) Examine the role of NO and nitrite in modulating heme-mediated lipid oxidation and antioxidant depletion in plasma from normal individuals and patients with SCO. 3) Examine the effect of nitrite on plasma lipid oxidation and arterial dysfunction in murine models of severe SCO and hereditary spherocytosis (HS). 4) Examine the effects of docosahexaenoic acid (an omega-3 fatty acid) on plasma lipid oxidation and vascular dysfunction in murine models of sever SCO and HS. Experiments will be conducted in in vitro systems, in systems using plasma from SCO individuals, and in mouse models of SCO and HS. The HS mice gave a severe hemolytic phenotype but exhibit different pathology than the SCO mice. We will use this comparison to tease apart the effects that are due to hemolysis alone and those which require additional aspects of the SCO phenotype. Specialist techniques that will be used include electron paramagnetic resonance spectroscopy, chemiluminescence based NO detection, polarographic oxygen measurements, liquid-chromatography and other analytical and histological techniques. Experiments will explore the role of hemolysis in SCO and the consequences of dietary intervention with respect to both the NO and lipid oxidation axis of the hypothesis. It is anticipated that data derived from these studies will provide valuable information with regard to future treatments of SCD.